Digital microfluidic platform for radiochemistry

ABSTRACT

Disclosed herein are methods of performing microchemical reactions and electro-wetting-on-dielectric devices (EWOD devices) for use in performing those reactions. These devices and method are particularly suited for preparing radiochemical compounds, particularly compounds containing  18 F.

RELATED APPLICATIONS

This Application is a continuation of U.S. patent application Ser. No.14,643,151, filed Mar. 10, 2015, now U.S. Pat. No. 9,193,640, which is acontinuation of U.S. patent application Ser. No. 13/500,785, filed Apr.6, 2012, now U.S. Pat. No. 9,005,544, which claims priority to U.S.National Stage filing under 35 U.S.C. §371 of International ApplicationNo. PCT/US2010/002756, filed Oct. 15, 2010, which claims priority toU.S. Provisional Patent Application No. 61/252,095 filed on Oct. 15,2009. The contents of the aforementioned applications are herebyincorporated herein by reference in their entirety. Priority to theaforementioned applications are hereby expressly claimed in accordancewith 35 U.S.C. §§119, 120, 365 and 371 and any other applicablestatutes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under CA119347 andRR020070, awarded by the National Institutes of Health and DE-SC0005056,awarded by the United States Department of Energy. The Government hascertain rights in the invention.

The use of microfluidic devices for performing chemical reactions,particularly radiochemical reactions and more particularly theproduction of tracers for positron emission tomography (PET) isdescribed in A. M. Elizarov, “Microreactors for RadiopharmaceuticalSynthesis,” Lab on a Chip, vol. 9, no. 10, pp. 1326-1333, May, 2009;C.-C. Lee, G. Sui, A. Elizarov et al., “Multistep Synthesis of aRadiolabeled Imaging Probe Using Integrated Microfluidics,” Science,vol. 310, no. 5755, pp. 1793-1796, Dec. 16, 2005, 2005 and A. Helene,“Positron Emission Tomography (PET) and Microfluidic Devices: ABreakthrough on the Microscale?,” Angewandte Chemie InternationalEdition, vol. 46, no. 11, pp. 1772-1775 (2007), all of which areincorporated in their entirety by reference herein. Microfluidic devicesprovide the option of reducing reagent usage, increasing control overreactions (e.g. heating and mixing), reducing overall system size, whenused in radiochemical synthesis, reducing radiation shielding needs, andthey provide practical routes to generic devices that can synthesize avariety of different radiolabeled probes on demand. Described herein isa novel radiosynthesis platform based on handling of fluids in dropletforms. The unique platform is demonstrated through the use of atechnique referred to as electro-wetting-on-dielectric (EWOD) which is avoltage-based actuation method to manipulate liquids.

Particular advantages of EWOD devices for radiochemical synthesisinclude, but are not limited to the following:

-   -   Inert materials. The wetted materials are typically only        fluoropolymers (e.g. Cytop™ or Teflon AF™). These materials have        high chemical and thermal stability and are expected to be        compatible with a wide range of reaction conditions.    -   Open channels. Open channels permit rapid evaporation of        solvents. Evaporation is a frequent operation during        ¹⁸F-radiosyntheses, wherein drying of [¹⁸F]fluoride ion is a        usual step, as well as removal of solvent prior to a        solvent-exchange operation. The open channels also enhance        mixing, which is restricted in other types of microfluidics        involving closed channels. Speeding up such operations is        important when working with short-lived radioisotopes.    -   Flexible fluid path. In principle, a single chip design with an        “array” of electrodes could perform a wide variety of syntheses        simply by reprogramming the droplet movements for different        syntheses. The single chip would drive down manufacturing costs,        and provide expandability to new probes with only a software        change. The flexibility could also be used to implement reaction        optimization, study of reaction kinetics, etc.    -   Liquid diversity. Small volumes of many different types of        liquids, including aqueous solutions and organic solvents, can        be manipulated within the same EWOD device. (Different        electrical signals may be necessary with some liquid        combinations.) In channel microfluidics, manipulation of small        volumes requires proper matching of liquids and channel surface        properties to avoid large losses; thus, to manipulate different        solvents in the same device (as is typical for radiochemical        reactions) requires specialized fabrication methods, including        hybrid devices based on multiple materials and locally treated        channel surfaces.    -   Straightforward fabrication. The EWOD chip is fabricated using        known integrated-circuit processing steps. For example,        electrodes are patterned using electrically conductive        materials, such as indium tin oxide (ITO) or gold on an        insulating layer, such as silicon nitride, the structure being        covered by a hydrophobic film. Chips can also be made using        industry standard printed-circuit-board manufacturing (plus        hydrophobic coating). The device simplicity allows many other        fabrication options, e.g., screen printing, inkjet printing, and        roll-to-roll printing, for a range of different cost and        performance. EWOD chips can be mass produced at a lower cost per        chip for disposable use.    -   Simple system. The EWOD device is operated only by using voltage        signals, typically below 100 V. A pressure source (e.g., pump),        flow regulator (e.g., valve), complex plumbing, moving parts,        high voltage (e.g., kV for electrokinetc drive), or high power        (e.g., thermal, electromagnetic) are not required. Consisting        only of a small printed circuit board, the complete system is        small and easy to fit into the hot cell, mini cell, or miniature        radiation shielded enclosure.    -   Small fluid volumes. EWOD devices can reliably manipulate fluid        volumes from hundreds of microliters to picoliters. Working at a        volume scale below the milliliter volumes of conventional        macroscale approaches enables faster processes (heating,        evaporation, and cooling, etc.), higher radioisotope        concentrations, lower amounts of reagents which are an important        source of undesired impurities, etc.

Other advantages include the capability of printing “dried” reagentsdirectly on an EWOD chip. Fresh-solvent droplets are used to redissolvethese reagents and use them in the chemical synthesis process. The useof reagents dried on the chip allows the construction of a “kit” forsynthesizing a particular probe—this has enormous potential forsimplifying probe synthesis, in a highly reliability and reproduciblemanner by the use of standard reagents of controlled quality.

As described herein, it can now be shown that liquid actuation on EWODscan be accomplished using transport of droplets, merging of droplets,mixing of droplets, or splitting of droplets. Droplets can be dispensedwith fixed or controlled volume (via capacitance or other feedback).Further, sensors and actuators can be integrated into chips, for examplefor liquid detection (monitoring processes) volume measurement, andtemperature control (heating and resistance feedback).

BACKGROUND

Microfluidic devices have been used in radiochemistry since about 2004.A recent review by Elizarov (ibid.) summarizes the various approachesthat have been taken. It is a common view in the field that the use ofmicrofluidics has many advantages over the predominant macroscopicmethods (manual and automated systems) for the production of PETtracers.

In regard to microfluidics, most researchers have used continuous flowmicroreactors (Elizarov (ibid.)) that still require substantialauxiliary equipment such as syringe pumps, F-18 drying subsystems, etc.As a result, these systems are bulky, complex, and expensive. The few“integrated” approaches (C.-C. Lee, G. Sui, A. Elizarov et al.,“Multistep Synthesis of a Radiolabeled Imaging Probe Using IntegratedMicrofluidics,” Science, vol. 310, no. 5755, pp. 1793-1796, Dec. 16,2005) that have been tried suffer from material compatibility problems.For example, PDMS is not compatible with many organic solvents and otherreaction conditions used in probe syntheses. Further, very lowreliability of operation has been observed. Integrated approaches alluse microfluidic devices with well-defined channel patterns. Thisrestricts flexibility and in most cases, requires that different probesuse different chip designs.

SUMMARY

Shown herein are improved, unique EWOD devices, reactant deliverysystems and processes for preparing radio-compounds using these devicesand delivery system.

Disclosed herein are methods of performing microchemical reactions andelectro-wetting-on-dielectric devices (EWOD devices) for use inperforming those reactions.

The device comprises:

-   -   a first substrate having one or more fluid paths on a first        surface thereof, each fluid path comprising multiple discrete        electrically conductive electrode pads spaced from and insulated        from the fluid path, adjacent conductive pads in said fluid path        separated from each other by non-conductive spaces, each of the        multiple conductive pads having electrically conductive lines        attached thereto, each conductive line provided for delivering        electrical signal to the conductive pad to which said conductive        line is attached to provide electrically directed movement of a        fluid droplet along said fluid paths,    -   one or more fluid delivery sites located on or adjacent the one        or more fluid paths, and    -   one or more reaction sites or heater sites located along the        fluid paths on said first substrate,    -   said fluid paths leading from the one or more fluid delivery        points to the one or more reaction sites or heater sites.

The device can also include a second substrate positioned over the firstsubstrate, the second substrate being position parallel to the firstsubstrate with a defined space between the first substrate and thesecond substrate. The second substrate can be electrically conductive orhave an electrically conductive film coating on a surface of the secondsubstrate facing the defined space, said conductive substrate orelectrically conductive film coating providing an electrical ground. Inone embodiment one or more portions of the first substrate extend beyondone or more edges of the second substrate to provide the one or morefluid delivery sites, the fluid delivery sites being located on oradjacent conductive fluid paths at the one or more edges of the secondsubstrate.

The heater sites can comprise, discrete electrically conductive pads,each discrete electrically conductive pad being independently connectedfor independent control of the temperature of each heater pad. Further,the one or more heater sites can comprise concentric, discreteelectrically conductive pads.

A dielectric layer and a hydrophobic film coating preferably cover atleast the conductive pads and conductive lines.

Also disclosed is a fluid delivery device for providing droplets ofreactants to the fluid delivery sites comprising:

-   -   a sealed container for holding liquid for delivery comprising        the chemical in a liquid carrier, said container having means        for continuously or periodically delivering a pressurized gas to        a head space in the container above the liquid for delivery,    -   a tube extending upward from a top portion of the sealed        container for transferring the chemical in the liquid carrier        from the sealed container to a delivery location positioned        above the sealed container, and    -   a source of pressurized gas and means for delivering said        pressurized gas to the sealed container in a controlled        continuous or periodic manner sufficient to prevent flooding.

Sensing means for detecting that a droplet of the liquid carrier hasreached the delivery location or an intended second location, can beprovided, said sensing means or a second sensing means also detectingthat no more than a desired quantity of the liquid carrier has reachedthe intended second location.

A procedure for using the above described electro-wetting-on-dielectricdevice (EWOD) comprises:

-   -   placing a droplet comprising a first chemical reactant in a        liquid carrier, preferably volatile, at one of the fluid        delivery sites, said delivery site being at or spaced from the        one or more heater sites,    -   if the droplet is placed at a delivery site spaced from the        heater site, providing an electric field in a serial manner to        the adjacent conductive pads spaced from said fluid path to move        the droplet from the fluid delivery point to a heater site,    -   placing a droplet of a second chemical reactant in a liquid        carrier, preferably volatile at one of the fluid delivery sites,    -   providing an electric field in a serial manner to the adjacent        conductive blocks spaced from said fluid path to move the        droplet of the second reactant from the fluid delivery point to        the heater site, and    -   heating the combined first and second reactants to cause a        chemical reaction between said first and second reactants to        form a desired intermediate or end product, and    -   further reacting or recovering the desired intermediate or end        product.

The droplet of the first reactant is heated at the heater site topartially or fully remove the liquid carrier by evaporation beforeadding the droplet of the second chemical reactant. Additional dropletsof the first reactant or the liquid carrier can then be transported tothe heater site before addition of the droplet of the second chemicalreactant, said additional droplets of the first reactant beingtransported to the heater either before evaporation, after partialevaporation or after completing evaporation of prior delivered dropletsof the first reactant. The droplet of a second chemical reactant in aliquid carrier is then combined with the first reactant on the heatersite either before evaporation, after partial evaporation or aftercompleting evaporation of prior delivered droplets of the firstreactant. The first reactant in combination with the droplet or dropletsof the second reactants are then heated for a sufficient period of timeto react to provide the desired intermediate or end product.

Additional volatile liquid carrier can be transported to the heater siteduring the reaction to maintain the first and second reactants in aliquid environment, partial or total evaporation being effected aftersufficient time has elapsed to complete the desired reaction. As analternative, more than two reactants can be delivered to the heater sitefor preparing an end product.

In one alternative embodiment a droplet of a mixture of chemicalreactants in a liquid carrier is placed at one of the fluid deliverysites. The droplet is then moved to a heater site and is heated to causethe desired reaction to produce the intended intermediate or endproduct, and remove the volatile liquid carrier.

The above described devices and method are particularly suited forpreparing radiochemical compounds, particularly compounds containing¹⁸F.

DESCRIPTION OF FIGURES

FIG. 1 is a schematic diagram showing an embodiment of an EWOD chip withstacked components.

FIG. 2 is a schematic drawing showing the control electrode pattern ofan EWOD chip incorporating features of the invention.

FIGS. 3-5 are schematic diagrams of three different heater embodiments.

FIG. 6 is a schematic drawing showing an embodiment of an EWOD chip withtwo liquid feeds.

FIG. 7 is a schematic drawing similar to FIG. 6 also showing theelectrical leads and connection sites.

FIGS. 8-16 are schematic diagrams of nine successive steps in the usesof an EWOD chip incorporating features of the invention.

FIG. 17 illustrates a temperature calibration system used to calibratethe heater electrode designs of FIGS. 3-5.

FIG. 18 is a calibration curve for a particular heater design generatedusing the temperature calibration system of FIG. 17.

FIGS. 19 A-D show a series of stages in the evaporation of a droplet.

FIG. 20 is the chemical reaction used in the cold run example.

FIG. 21 shows the chemical reaction used in the hot run example toprepare [¹⁸F]FNB.

FIG. 22 is the TLC graph of the product from the hot run ([¹⁸F]FNBproduction)

FIG. 23 is the TLC graph showing the results of [¹⁸F]FDG production.

FIG. 24 is the TLC graph showing the results of [¹⁸F]TAG production.

FIG. 25 is a schematic diagram showing a second EWOD design.

FIG. 26 is an enlarged diagram of the concentric heater of FIG. 25.

FIG. 27 is s schematic drawing of a pneumatic delivery system.

FIG. 28 is a schematic diagram illustrating the positioning of fluid ina delivery tube using the system of FIG. 27.

FIG. 29 is a TLC graph showing the results of the production of apreclinical dose of [¹⁸F]FDC purified using a mini-cartridge.

FIG. 30 shows the chemical reaction for the preparation of1-(4-bromobutoxy) naphthalene.

FIG. 31 illustrates the TLC separation of 1-(4-bromobutoxy)naphthalene.

FIG. 32 is a schematic diagram of an EWOD control system for sensingdroplet volume.

FIG. 33 is a schematic diagram of a circuit used for theoreticalprediction of the relationship between voltage and capacitance(impedance).

FIG. 34 is a schematic diagram showing pipette delivery of a droplet toa loading position on the EWOD.

FIG. 35 shows the chemical reaction used in the hot run example toprepare [¹⁸F]FDG.

DETAILED DESCRIPTION

The structure of an EWOD chip 10 is shown in FIG. 1. The operation ofEWOD devices and the driving of fluids across the surface of thesedevices is described in Shih-Kang Fan, Peter Patrick deGuzman, Chang-JinKim, “EWOD Driving of Droplet on N×M Grid Using Single Layer ElectrodePatterns”, Solid-State Sensor, Actuator and Microsystems Workshop,Hilton Head Island, S.C., Jun. 2-6, 2002, said article incorporated inits entirety by reference herein. In a preferred embodiment, two glasssubstrates 12, 14 (a top plate and a bottom plate) are held together ina spaced apart orientation with a 100 μm spacer 16 (i.e., double-sidedtape) to form a gap 18 for placement of liquid droplets 20 (small dropsof a liquid). The bottom-plate substrate 14 has control electrodes 22,usually comprising ITO or gold, formed or positioned on the top surfacethereof. An insulating dielectric layer (such as silicon nitride) 24,located over the control electrode 22 is covered by a hydrophobic filmlayer 26, such as a Cytop® or Teflon® film. The bottom surface of thetop-plate substrate 12 is covered by a conductive film 28, such as ITO,which functions as a ground electrode. However, in alternativeembodiments the second substrate can be eliminated, providing an openstructure with the droplet resting on the first substrate upper surface.In such an instance the ground electrode can be located in a differentlocation, for example below or within the first substrate. In the twosubstrate structure, a layer of an insulating dielectric 30 (such assilicon nitride), which is preferably thinner than the insulatingdielectric 24 on the bottom plate, preferably one-tenth the area of thelower plate insulating dielectric 24, covers the ground electrode 28. Ahydrophobic layer 32 covers the lower face of the upper dielectric 30.EWOD chips can be formed using various known techniques including butnot limited to standard chip formation technology using printed circuitboard fabrication techniques or flexible electronics.

Preferentially, the EWOD chip 10 is transparent except for theelectrical connection pads 41 along two edges of the chip and severalconductive (preferably gold) connection lines 52 connected to thecontrol electrode pathways 42 leading to the heater 44. Droplets 20 ofwater, DMSO, MeCN, or other liquid carriers are transported into andalong the gap 18 between the two substrates 12, 14 along the pathways 42by voltage signals applied to the connection pads 41 and through theconductive connection lines 52.

An electrode pattern of an EWOD chip design 38 incorporating features ofthe invention is shown in FIG. 2. Located within the pattern are tworeservoirs 40 which may receive the solutions involved in the reaction.The electrical connection pads 41 are connected by conductive connectionlines 52 to the conductive pathways 42 (also shown as control electrodes22 in FIG. 1). The conductive pathways 42 comprise a series of discreteelectrically conductive pads separated by non-conductive spaces 43, asbest shown in FIGS. 6 and 8-16, below the surface of the chip. In theexample shown in the figures, 1 mm×1 mm squares of ITO. The centrallylocated square is a heater 44. FIGS. 3, 4 and 5 show three alternativeheater 44 designs. FIG. 3 is a simple heater electrode design 46provided to heat the solution droplet placed on the electrode 44. FIGS.4 and 5 are first and second versions of a self-centering heatingelectrodes 48, 50, which heat the droplet 20 centered on the heater.Shown at the bottom corners of the heater area are a ground lead 54 anda voltage lead 56 that are connected to a power supply (not shown).

FIG. 6 is a schematic drawing showing an embodiment in which a coverplate 58 covers only a portion of the EWOD chip 38. FIG. 7 is anotherview of the chip shown in FIG. 6 also showing the connection pads 41 andconductive connection lines 52. The incomplete coverage of the chip 38by the cover plate 58 creates edges preferably near at least onereservoir 40 so that reagents can be loaded into the gap 18 using apipet or injection needle 146 onto the EWOD chip (see FIG. 34). Theembodiment of FIGS. 6 and 7 shows two possible edge loading positions 60which permit separation of an aqueous reagent (for example an F-18solution) from an anhydrous solution (precursor solution) by usingdifferent pathways on the chip, thus avoiding cross-contamination due toresidue that is potentially left behind after droplet motion.Alternatively, the loading positions 60 can comprise holes formedthrough one of the substrates 12, 14 as shown in FIGS. 27 and 28.

Each connection pad 41 of an EWOD chip is supplied a DC or AC signal(e.g. frequency (˜10 KHz) and voltage (˜100V)). By applying voltages tovarious different connection pads 41 in sequence along the edge of thechip, droplets 36 deposited at the loading positions 60 can be caused tomove along pathways 42 along the chip surface. Instruments used to movethe droplet include a signal generator, high voltage amplifier, digitalI/O control box, a relay array and a source meter to supply power to theheating electrodes 48, 50 and to measure the temperature. Thesecommercially-available or custom instruments can be controlled bymanually or by software such as LabView software. One skilled in the artwill recognize that more than 2 reservoirs 40 and loading positions 60can be provided on the chip to provide delivery of 3 or more reactantsthrough different pathways 42.

Embodiments and features of EWOD devices incorporating features of theinvention that have been built and tested by applicant have included thefollowing:

-   -   Movability of solvents relevant to ¹⁸F-radiochemistry    -   Ability to evaporate droplets of water or acetonitrile (also        mixtures containing K₂CO₃ and K.2.2.2)    -   Redissolving the residue left on the heater after solvent        evaporation    -   Performing reactions at elevated temperatures in volatile        solvents    -   Cold synthesis of fluoronitrobenzene (evaluated with TLC)    -   Hot synthesis of [¹⁸F]fluoronitrobenzene (evaluated with        radio-TLC)    -   Hot synthesis of [¹⁸F]fluorodeoxyglucose (evaluated with        radio-TLC)    -   Synthesis of 1-(4-bromobutoxy) nophthalene

While frequency and voltage may vary depending on device configurations,preferred minimal frequency and voltage of the power supply formanipulating droplets of relevant solutions on the EWOD chip for aparticular configuration are shown in Table 1. K₂CO₃ solution (similarto composition of [¹⁸F]fluoride after elution from anion exchange resin)MeCN, DMF and DMSO (in which precursors of many reactions is dissolved)are preferred carrier liquids.

TABLE 1 Minimal frequency and voltage for liquid manipulation SolutionFrequency (KHz) Voltage (V) DMSO 15 90 DMF 20 82 Methanol 5 77.3 THF 1570 K2CO3 1 75

Several designs for heaters 44 were fabricated and tested. As a firststep, each electrode design was calibrated by immersion in an oil bath.FIG. 17 illustrates a calibration system 70 comprising a hot plate 72that heats the silicone oil 74 contained in a vessel 76. The resistanceof the heater 44 is measured with an ohmmeter 78 and oil temperature(assumed to be same as immersed EWOD device temperature) is measuredusing a thermocouple 80. Resistance is a function of temperature inaccordance with the formula R=R_(o)[1+α(T−T_(o)), where R is resistanceat temperature T, R_(o) is resistance at temperature T_(o), and a is thetemperature coefficient of resistance. The temperature coefficient ofresistance for ITO on the EWOD chip is determined by plotting T vs. Rand finding the slope of its linear fit (FIG. 18).

With a known temperature coefficient of resistance for the depositedITO, the temperature of heater 44 can be controlled on the EWOD chip 38.AC or DC current can be applied through positive and negative leadsattached to the resistor (heater 44) to heat it, while its resistance ismeasured. When the calculated resistance for a desired temperature ismet, that temperature is presumed to have been reached. The temperatureis maintained at the desired set point by software PID control over thecontrol circuit.

The heater 44, the EWOD control electrodes 22 comprising the conductivepathway 42 and EWOD connection lines 52 on a preferred embodiment of theEWOD chip are made of ITO. However, the connection line from the controlcircuit (not shown) to the heating electrode 44 is made from gold toprevent the connection line from heating when current is applied becausegold is a much better conductor than ITO. The heater 44 can reachtemperatures greater than 200° C. without damage, but the hydrophobiclayers should not be heated above about 190° C.

To test evaporation using the above described embodiment about 400 nL of1M K₂CO₃ solution was transported, using the techniques describedherein, to the heater 44 location and the temperature was raised to 105°C. The majority of the solvent was evaporated within 2 min. For lowerboiling point solutions (e.g. solutions with lower salt concentration,or solutions containing low-boiling-point organic solvents), evaporationtime is substantially lower.

EXAMPLE 1 Demonstration of Non-Radioactive (“Cold”) Synthesis

To demonstrate the basic capability for synthesis, non-radioactivefluoronitrobenzene was synthesized on the EWOD chip. This synthesisprocess on the EWOD chip is shown schematically in FIGS. 8-16 and thechemical reaction is shown in FIG. 20. As the reaction of fluoride withthe precursor of 1,4-dinitrobenzene is sensitive to water, this cold runprocedure effectively tests the completeness of water evaporation. Theproduct solution can then be separated by TLC and detected with UVlight. In the following description “SM” means “starting material”(unreacted precursor) and “P” means the sample of the product.

Two solutions were prepared to perform this reaction.

-   -   Solution 1 (Fluoride solution) was 115 mM KF+234 mM Kryptofix        K.2.2.2 in a solution of acetonitrile (MeCN) and water (88:12        v/v ratio), and    -   Solution 2 (Precursor solution) was 119 mM 1,4-dinitrobenzene in        DMSO.

Referring to FIGS. 8-16 and 34, reagent droplets 36 were loaded onto theEWOD chip 38 at the loading position 60 along the edge of the coverplate 58 using a pipette (FIG. 8) The EWOD chip electrodes thentransported the droplet along the pathway 42 to the heater 44 (FIGS.8-9) where it was heated and the liquid evaporated. More particularly,the procedure using the EWOD chip is as follows:

-   -   1. 500 nL of a first solution was placed at the edge inlet 60        and transported to the heater 44    -   2. The heater 44 was heated to 105° C. for 3 minutes. (FIG. 10)        leaving a dried residue 82 on the heater location    -   3. 600 nL of MeCN was loaded at edge inlet 60 (FIG. 11) and        transported to the heating electrode 44 (FIG. 12).    -   4. The MeCN was then heated at 105° C. using the heater for 3        minutes (FIG. 13) to evaporate the MeCN to help remove residual        water from the residue 82. FIG. 19A schematically shows the        droplet located at the heater 44 position, the arrows        representing the release of vaporized liquid carrier. FIG. 19B        shows partial condensation 62 of the vapor on the bottom of the        upper plate 12. FIG. 19C shows dissipation of the condensed        droplets as the lower surface gets warmer as a result of heat        from the heater. FIG. 19d shows complete or substantially total        evaporation of the liquid from the droplet, leaving behind the        dried solute residue 82 of [¹⁸F] compound    -   5. Step 3 and step 4 were repeated several times. (This process        is called azeotropic distillation and its purpose is to remove        as much residual water as possible due to water-sensitivity of        fluorination reaction.) A positive correlation was observed        between the number of times steps 3-4 are repeated and the end        product yields.    -   6. 500 nL of a second solution (precursor) was then loaded at        the second edge inlet 60 (FIG. 14) and transported to the        heating electrode 44. (FIG. 15).    -   7. The combination of dried, water free first solution residue        and precursor were then heated to 120° C. using the heater for 3        minutes to react the two materials, generating the desired        reaction end product 84. (FIG. 16).

After the evaporation and reaction, the top-substrate andbottom-substrate of the EWOD chip were separated. The product wasremoved using a capillary, and then TLC separation and UV lightdetection was performed with buffer dichloromethane and hexane (1:1v/v). Through comparison of a sample of the product of on-chip reaction,along with “standards” (precursor and a “cold standard” of the knownfinal product) the proportion of desired product and unreacted precursorwas estimated to provide an estimate of the reaction yield.

EXAMPLE 2 Radiosyntheses (“Hot” Runs)

A corresponding hot run, i.e. radiosynthesis of [¹⁸F]fluoronitrobenzene([¹⁸F]FNB) was also conducted. The hot run reaction procedure wassimilar to the cold run, except using [¹⁸F]fluoride produced in aacyclotron rather than KF solution. No-carrier-added [¹⁸F]fluoride ionwas produced by 11 MeV proton bombardment of 98% enriched [¹⁸O]water ina silver target body using a RDS-112 cyclotron. Two reagent solutionswere prepared:

Solution 1 (F-18 solution) contains 55 mM K₂CO₃, 112 mM KryptofixK.2.2.2, and 58 nM F-18 in a mixture of MeCN and water in the ratio84:16 (v/v);

Solution 2 (precursor solution) contains 119 mM 1,4-dinitrobenzene inDMSO.

As is typical of ¹⁸F-radiosyntheses using [¹⁸F]fluoride, an importantstep was drying and activating the F-18 after its elution from ionexchange cartridge. Reaction of the K¹⁸F/K_(2.2.2) with the precursor(1,4-dinitrobenzene, typically in DMSO) then yields the product1-[¹⁸F]fluoro,4-nitrobenzene. The chemical reaction is shown in FIG. 21.The detailed steps of the hot run were identical to that for the coldrun.

Solution 1 was deposited at the loading position on the chip,transported to the heater location and evaporated and solution 2 wasthen brought to the same location. Following the synthesis, the productwas separate by silica gel TLC (buffer MeCN/H2O 95:5 v/v) and visualizedwith a radio-TLC scanner to determine the relatively amounts ofradioactive species and thus the reaction yield.

See FIG. 22 for the TLC profile.

Using the radio-TLC profile, the area of each peak is integrated. Fromstudies of “standards” it is known at which positions differentchemicals will remain under the conditions used for separation. In theexample of FIG. 22, the first (left) peak corresponds to unreacted[¹⁸F]fluoride. The second (right) peak corresponds to the desiredproduct. The TLC result shown in FIG. 22 indicates that the conversionefficiency (from F-18 to desired product [¹⁸F]fluoronitrobenzene) wasabout 96% Conversion was consistently high for several runs with anaverage conversion efficiency of 78%±12% (n=8 runs), consistent withconventional synthesis of this compound. The sample recovery was about29% as discussed above. Thus radiochemical yield (RCY) of[¹⁸F]fluronitrobenzene from [¹⁸F]fluoride in the EWOD chip is calculatedto be 96%×29% (i.e., 28%). Varying operating conditions and handling ofthe materials has since resulted in far lower losses with recoveries inthe range 80-100%.

A hot run was also performed to produce ¹⁸F-labeled fluorodeoxyglucose([¹⁸F]FDG). According to the reaction scheme shown in FIG. 35. The hotrun procedure for [¹⁸]FDG is similar to that for[¹⁸F]fluoronitrobenzene, with a few differences. The main difference isthat [¹⁸F]FDG requires a second reaction, a hydrolysis step—to removeacetyl protecting groups from the fluorinated intermediate. Also, thenucleophilic substitution of ¹⁸F onto the precursor occurs in thesolvent MeCN instead of DMSO.

Reaction of the K¹⁸F/K2.2.2 activated complex with precursor mannosetriflate (typically in MeCN) yields the intermediate [¹⁸F]FTAG. The MeCNis then evaporated and replaced by HCl for hydrolysis to form the finalproduct, [¹⁸F]FDG. Three solutions were prepared:

-   -   Solution 1 (F-18 solution) contains 55 mM K₂CO₃, 113 mM        Kryptofix K.2.2.2 and about 26 nM [¹⁸F]fluoride in a mixture of        MeCN and water (84:16 v/v).    -   Solution 2 (precursor solution) contains 114 mM mannose triflate        in MeCN.    -   Solution 3 (deprotection solution) contains 1N HCl.

For radiosynthesis of [¹⁸F]FDG, a 500 nL droplet of Solution 1 wasloaded onto the EWOD chip and moved to the heater site, where it washeated at 110° C. for 3 minutes leaving a residue. An azeotropic dryingstep was then repeated several times (generally 3-5; however, as many as10 repeats have been used). The azeotropic drying step consisted ofloading a 600 nL droplet of MeCN onto the chip, moving it to the heaterto dissolve residue remaining from the previous step, and drying at 110°C. for 3 minutes. Once drying of the fluoride was complete, a 900 nLdroplet of Solution 2 was loaded onto the chip and moved to the heatersite. Heating at 85° C. for 5 minutes formed the [¹⁸F]FTAG intermediate.Because MeCN has a low vapor pressure and rapidly evaporates during thereaction, 600 nL droplets of MeCN were added every 30 seconds and movedto the heater to ensure a liquid-state reaction. The [¹⁸F]FTAG dropletwas dried (MeCN evaporated) by heating it to 95° C. for 1 min. 1 μL ofHCl (1 M) was loaded, moved to the [¹⁸F]FTAG site, and heated at 95° C.for 3 minutes to perform hydrolysis for the removal of a protectiveacetyl group and form [¹⁸F]FDG.

The sample was recovered from the EWOD chip and separated by silica TLC,MeCN and water (95:5 v/v). The resultant separated materials weremeasured with a radio TLC reader (FIG. 23).

FIG. 23 shows the composition of a sample recovered by dissolving into awater solution of [¹⁸F]FDG synthesized on the EWOD chip.Radiochemically, it is primarily FDG. FIG. 24 shows the composition of asample recovered by dissolving into a MeOH solution of [¹⁸F]FTAGsynthesized on the EWOD chip.

Several repetitions of [¹⁸F]fluoronitrobenzene synthesis and [¹⁸F]FDGsynthesis have been performed with high repeatability. Synthesis ofadditional relevant probes, includes[18F]fluoro-3′-Deoxy-3′-L-fluorothymidine [¹⁸F]FLT,9-[4-[18F]fluoro-3-(hydroxymethyl) butyl]guanine [¹⁸F]FHBG, and3,4-dihydroxy-6-[18F]fluoro-L-phenylalanine [¹⁸F]FDOPA

Alternative approaches within the scope of the invention include:

-   -   A microfluidic chip (e.g. PDMS) to deliver small reagent volumes    -   A robotic pipetting system    -   Connection of tubing-to-the EWOD interface for delivery of        reagents    -   Automated methods to recover the final product    -   Holes through the chip cover plate for use as loading positions    -   Use of tubing, pipette, or vacuum to recover liquid    -   A standalone “radiosynthesizer” based on an EWOD chip, complete        with reagent loading system, product collection system,        monitoring system, control system, and radiation shielding.    -   Design of computer-controlled system for simple reaction        optimization/exploration (including varying temperature,        concentrations, mixing ratio, reaction time, etc. . . . )    -   Integration of EWOD chip with radioactivity camera to study        surface adsorption and track fate of all radioactivity on the        chip (to determine yields, and losses that can be optimized)    -   Added channels inside the EWOD chip in select locations.    -   Limitation or prevention of evaporation to allow storage of        chemicals in reservoirs on the chip.    -   Isolation of water vapor from dry organic solvents on chip    -   Walls on the chip to help with elution off of soiled electrodes        (e.g. with gas or liquid)    -   Inclusion of Argon or Helium filled chambers to protect air or        water-sensitive reagents    -   On-chip storage of dried reagents. This may include depositing        liquid reagents, drying them (evaporation), or reconstituting at        time of synthesis by addition of droplets of pure solvents. By        having reagent pre-loaded, the chip will be much easier to use        by individuals not trained in radiochemistry.

In the designs above, 1 mm by 1 mm square resistive elements were usedfor heating droplets to evaporation and reaction temperatures. With agap of 100 μm (approximately 0.2 mm) between the cover plate and chip,the nominal droplet volume of about 100 nL allows processing of up toseveral hundred nanoliter droplets. At this length and volume scale,feedback control using simple measurements of the average temperatureover the whole resistive element can maintain a controlled temperaturedistribution over the small area resistors. However, the small arealimited the reaction volume and amount of starting material that couldbe processed.

While the above examples describe the delivery of first and seconddroplets of two reactants, it is also contemplated that the first andsecond reactants can be mixed together prior to delivery and then adroplet of the combined reactants can be placed at the delivery site formovement to the heater, or placed directly on the heater where thereaction is performed. As a further alternative droplets of additionalreactants can be placed and moved to the same heater site for use in thechemical reaction.

The use of an EWOD chip 98 incorporating larger circular heaters 100having 12 mm overall diameter such as shown in the further chipembodiment shown in FIG. 25, and in the enlarged view of the heater 100enclosed in the circle 26 of FIG. 25 (FIG. 26), a larger amount ofproduct can be generated with each heater 100 (approximately 23 μL). Inorder to avoid disparate hot and cold regions when only the averagetemperature over the entire large heating area remains controlled, theheater is divided into 4 concentric resistive elements 102, 104, 106,108, each with its own feedback control of temperature (not shown).Compared to the first described heater design 44, such as describedabove, this multi-element heater can center the droplet and maintain itstemperature more uniformly as the droplet shrinks during evaporation.

The larger size of the heater 100 enables processing to produce largeramounts of tracers. Also, when using volatile solvents, the largervolumes slightly prolongs the droplet lifetime at a given temperature,thereby allowing longer duration reactions, or requiring less frequentreplenishment of the evaporating solvent.

A multichannel heater controller and driver (not shown) provides anexpandable platform for achieving and maintaining multiple, independentEWOD reaction sites at precise temperatures. The instrument wasspecifically designed for the resistive micro-heaters patterned on theEWOD chip. It incorporates a low noise, real-time, zero resistancecurrent measurement and amplification while providing a self-powered,amplified heater driver with parameters which can be controlled bydedicated software. The platform enables parallel reactions atindependent temperatures on the same or separate EWOD chip as wellprecise spatially varying temperature profiles for a single reactionsite, when used in conjunction with concentric heaters 100 or resistiveelements patterned in close spatial proximity.

The first embodiment discussed above used an expensive source meter toprovide the measurements for feedback control. Advantages of thetemperature control circuit in the second EWOD embodiment 98 include:

-   -   No measurement interference with the heater electrodes on the        EWOD chip. The system introduces negligible resistance and        current while making measurement    -   The system can make measurements and control multiple heated        reaction sites independently    -   The system allows high speed measurement and control    -   The system provides amplification to interface with readily        available data-acquisition systems for precision temperature        control and measurement    -   The software is designed to calibrate a new chip, by making        automatic initial measurements, such as resistances at room        temperature and making adjustments for offset currents    -   The software incorporates digital low-pass filters and can make        measurements at up to 20,000 samples/sec from each independent        heater to achieve near real-time measurement and control to        provide further stability by averaging    -   Circuit size and cost are small

Preferred operating conditions for loading fluid solutions containingsamples, reagents, catalysts etc. onto the chip for EWOD-basedradiochemistry system are:

-   -   Delivery of small droplet volumes (typically 0.1 to 2 μL)    -   Delivering reagents rapidly and frequently to EWOD chip (e.g. to        replenish evaporating solvents)    -   Compatibility with wide variety of reagents (solvents, corrosive        agents, etc.) over long time periods (e.g. so that reagents        could be supplied in a “kit”)    -   Disposability to avoid the need for washing to eliminate        cross-contamination, and to enable rapid removal and setup of        reagent kits for ease of use

Referring to FIG. 27, a pneumatically-driven fluidic loading system 120is shown. It utilizes inexpensive/disposable components in contact withthe reagents, while the more expensive actuating components such asvalves, pressure regulators etc. do not contact the reagents. Thispneumatically driven fluidic actuation is used for loading liquids intothe microfluidic system. In one embodiment, fluid (liquid reagent) 122is delivered from a supply vessel 124, such as a sealed chamber (e.g.vial). Two tubes are inserted into the vial. A first tube 126 is usedfor delivering an inert gas 128 to the space above the liquid; thesecond tube 130, which has one end immersed into the liquid reagent 122,is used for delivering the reagent. When pressurized inert gas 128 isdelivered to the vessel 124 it pressurizes the head space 132 in thesealed vessel 124, thus pushing liquid out of the second tube (thedelivery tube) 130.

As a method of controlling over-delivery of fluids which can occur withpneumatic delivery systems, the added features described below can beused to deliver small volumes of fluids without flooding. As shown inFIG. 27, an EWOD device is positioned (vertically) above the deliverytube 130 which is inserted into an EWOD loading position 60 on one endand which connects to the reagent vial at its other end. While theentire loading system 120 is shown in its preferential orientation, itis not necessary that it all be vertically disposed below the EWOD. Toreach the EWOD device from the reagent vial, the fluid must travel atleast some vertical distance upwards. The reason for such an arrangementis that the downward gravitational force on the liquid column directlybelow the EWOD inlet port provides a counteracting force that opposesthe surface tension force that tends to flood the device. The Gasdriving pressure is provided to the reagent vial 124 via a valve and apressure regulator (not shown). Capacitive sensing 134 over the actuatedEWOD electrodes is used to detect when the fluid has reached the device.An additional liquid sensor 136 can be provided to confirm the rise ofthe fluid meniscus 138. FIG. 27 shows the sensor 136 positioned adistance from the EWOD; it can be positioned closer to the EWOD or thedelivery system can be operated without the sensor 136.

Another feature of the delivery systems is that instead of applying asteady pressure into the reagent vial 124, pulses of pressure areapplied. The pulses of pressure can be applied either using one or morepneumatic control components such as pumps, valves, pressure regulators,flow controllers, or a combination thereof. When a sufficiently highpressure pulse is applied, the liquid in the tubing rises, with thehigher pressure force counteracting the downward gravitational force toproduce a net upward force. When the pressure is turned off (vented orreduced to a lower pressure), the gravitational force produces a netdownward force, decelerating the meniscus 138 rise in the tubing andeventually causing it to fall. (It should be noted that while thecapillary force would also figure in the force balance, it would remainconstant during the meniscus 138 rise or fall in a uniform cross-sectiontubing and therefore simply be another offset of the gravitationalforce.) By oscillating the pressure, the meniscus 138 can be kept closeto the chip, but with oscillating position so that the fluid momentumalways remains low in either direction (toward or away from chip). Thisoperation is illustrated in FIG. 28.

Another important feature of the technique is the sensing of the dropletat the EWOD device, prior to reaching the EWOD 98 or positioned on theheater 100. Several mechanisms could be used for the sensing, e.g.electrical, chemical, electrochemical, thermal, optical etc. or acombination thereof. As soon as a sufficient amount of liquid is sensedto be positioned on the EWOD device, the pressure is dropped, allowinggravity to pull the liquid back. Since the momentum of the rising liquidwas not allowed to build up beyond one pulse width, the overshoot involume, if any, is limited and flooding is avoided. EWOD actuation canbe turned on over some or all the wetted region on the device in orderto hold some of the reagent fluid on the EWOD device, while the rest ofthe fluid is pulled back. In order to improve the accuracy of dropletvolume dispensed, a secondary on-chip droplet dispenser can be used tocreate droplets from the loaded volume.

There is inherent robustness in this approach, which is tolerant ofvariations of the pneumatic control, as well as the properties, such aswetting properties, of the liquid being delivered and surfacecharacteristics of the materials used in fabrication of the EWOD.Firstly, a system relying purely on pneumatic control has limitedprecision over the volume of liquid dispensed. Due to thecompressibility of the inert gas 128 in the system, there can be asignificant lag between the time the pressure control valve is actuatedand the time the pressure actually changes at the surface of the fluid.The momentum built up during the fluid movement prior to pneumatic valveclosure (when attempting to “stop” the flow) can lead to “overshoot”during this lag time. While this effect can be avoided by the use of aslow flow rate to nudge the fluid flow slowly forward, this is often notpractical in terms of factors such as time required and precision ofpressure regulation, as well as phenomena like contact angle hysteresisand contact line pinning at the leading edge. Additionally, thepossibility of uncontrolled fluid flow is further amplified fornon-aqueous solutions often required in chemical synthesis, becausethese materials can be more wetting of the EWOD device than water.Following introduction of the droplet into the EWOD device, as thedroplet volume increases, a wetting droplet experiences a greatersurface tension force (proportional to the interfacial length) whichresults in the liquid being pulled into the chip, thus providing apositive feedback that could lead to a runaway volume delivered to thechip (a process referred to as flooding). As a result, many previouslyavailable microfluidic systems employ on-chip or off-chip liquid valvesto achieve greater precision over start and stop of liquid flow.However, on-chip valves are not available in EWOD systems, and off-chipliquid valves are expensive as well as not disposable, and would requirecleaning after each use.

It is therefore desirable to automate the delivery of reagents to theEWOD chip to increase ease of use and to protect the operator fromexposure to radiation. Many methods exist for pumping liquid from areagent reservoir to the chip. The challenge is determine when theinitial gas has been removed from the delivery system and when theliquid arrives at the desired location. While accurate metering pumpsand knowledge of the system geometry can be beneficial in improvingdelivery control, the use of volatile solvents also introducesuncertainty into the exact position of the liquid meniscus and thedroplet size that reaches the heater position. Evaporation occurs fromthe end of the needle/tubing/etc. into the open space of the EWOD chip;a significant fraction of a unit-droplet-volume can evaporate withinminutes. Thus, real-time sensing of liquids is desirable.

It has been suggested by others that liquid sensing can be accomplishedin EWOD chips by measuring capacitance, but these techniques alsorequire optical observation using a high resolution CCD camera orexpensive capacitance meters. Disclosed herein is a capacitive sensingtechnique that uses greatly simplified hardware and software. Thisreal-time liquid sensing system is designed to sense, quantify andidentify liquids on the EWOD chip without requiring additional hardware,other than a single resistor, to be added to the EWOD chip or controlsystem that is already used. The measurement is performed using ACsignals and a phase-locked loop technique to extract the measured signalwith high signal-to-noise ratio and a volume accuracy as small aspicoliters.

In the EWOD control system 140, such as shown in FIGS. 32 and 33, asignal generator 142 and amplifier 144 generates a high voltage(100-200V) signal (1-20 kHz). Individually addressable relays apply thisvoltage selectively to the desired electrodes to move the fluid. Toperform sensing in the feedback system, one small resistor 146(approximately 1 kΩ) is added at the ground line in controlling circuitand the voltage is measured across the resistor instead of directlymeasuring the capacitance (FIG. 32). This resistor does not appreciablychange the voltage across the liquid. To perform a capacitancemeasurement, the EWOD electrode is activated (with AC signal) at thesite where measurement is desired. The out of phase voltage signalacross the external resistor is measured via an analog-to-digitalconverter to determine the volume of liquid at the site of the activeEWOD electrode. A phase locked loop (using unamplified sinusoidal signalas a reference) extracts the desired signal. Approximating the system asa parallel plate capacitor (see FIG. 33), it can be shown that thesignal is a constant plus a term that is linearly proportional to thefraction of the electrode area covered with liquid, from which volumecan be determined as it is linearly proportional to the dielectricconstant minus one (which depends on the liquid), when the electrodesize and gap spacing are known. Data collected for droplets of water andDMSO of different volumes (measured via CCD imaging), confirm the linearrelationship. Once the electrode is completely covered with liquid, thesignal saturates and a further increase in droplet volume cannot bedetected.

If it is desired to measure capacitance at more than one sitesimultaneously, instead of inserting a single resistance on the groundline, one can instead insert resistors on the desired individualhigh-voltage electrode lines.

In summary, advantages of this approach for monitoring reagent loadingto an EWOD chip, include, but are not limited to:

-   -   Minimal hardware addition to the EWOD system (A single precision        resistor)    -   The measurement of voltage is performed at one point in the        circuit for all electrodes, while liquid sensing selectivity        spatial precision is provided by EWOD's normal operating        condition of activating a single electrode at a time    -   The system requires no additional signal generation for        measurement, as the frequency specific measurement is provided        by the applied AC potential used for normal EWOD operation    -   The measurement is based on the lock-in amplification technique,        which essentially filters out all noise outside a narrow        frequency band of interest. As such the system is immune to        major contributing noise factors such as drifts, 60 Hz noise and        ground loops    -   The low noise design allows accurate measurement of liquid        volumes onto the EWOD chip as small as a pico-liter using        readily available 16 bit digitizers and without incorporating        low noise amplification    -   The systems responsiveness (as fast as 1 ms for 10 nl accuracy)        and accuracy (down to 1 pico-liter for 100 ms response time) can        easily be adjusted using software    -   The software can very quickly indicate the presence of liquid,        which has many practical applications for chemical reactions        performed on EWOD, and then refine its accuracy of volume        measurement and liquid identification as needed and depending on        time available.

Described above are methods for synthesis of [¹⁸F]FNB and [¹⁸F]FDG. Inboth cases, there are several steps where droplets are completely dried,i.e., solvent evaporated, before proceeding with the next step. However,if all the liquid is allowed to evaporate, the heat conduction path fromthe bottom of the chip assembly, where heater is located, to the top ofthe chip assembly (i.e., the cover plate 58) is broken. As a result thetop is substantially cooler than the controlled temperature. Any solidor liquid residue on the top portion will thus experience a differenttemperature profile than that on the bottom. This can lead to someunreliability of processes such as [¹⁸F]fluoride drying.

To ensure a more uniform and controllable temperature, the synthesisprocess was modified so that complete evaporation never occurs. Forexample, during the process of drying the [¹⁸F]fluoride, new droplets ofMeCN were continually added (which were then transported to the heatersite) to replenish what was evaporating. In one example, fresh dropletswere loaded every 10-20 seconds for 5 min. Radiosynthesis of [¹⁸F]FDGand [¹⁸F]FTAG was successfully performed with yields comparable toconventional synthesis approaches, suggesting that this modified dryingprocess was successful at removing traces of water and ensuring areactive [¹⁸F]fluoride complex.

EXAMPLE 3

Materials and Characterization: Anhydrous acetonitrile (MeCN, 99.8%),anhydrous dimethyl sulfoxide (DMSO, 99.9%), potassium carbonate (99%),mannose triflate for PET imaging and 4,7,13,16,21,24,-hexaoxa-1,10,diazobicyclo(8.8.8) hexacosane 98% (K 2.2.2) were obtained fromSigma-Aldrich and used as received without further purification. 1 N HCl(certified, Fisher Chemicals) were purchased from Fisher Scientific andused as received. Synthesis was performed with EWOD chip 98 having 4concentric circular heater electrodes 102, 104, 106, 108 (FIGS. 25, 26).

Synthesis: No-carrier-added [¹⁸F]fluoride ion was generated byirradiation of 97% ¹⁸O-enriched water with an 11 MeV proton beam usingan RDS-112 cyclotron (Siemens). 50 μL (20 mCi) of the aqueous[¹⁸F]fluoride ion was added to a 40 μL mixture of K 2.2.2 (26 mM) andK₂CO₃ (7 mM) in MeCN:H₂O (98:2 v/v). Mannose triflate (5 mg; 0.01mmoles) was dissolved in anhydrous DMSO (100 μL) to obtain aconcentration of 104 mM. The [¹⁸F]fluoride mixture (2 μL) was pipettedonto the EWOD chip 98 through a dedicated loading position 60 at theedge of the cover plate 58 and transported to the heater 100 viaelectrowetting forces. This loading process was repeated 3 additionaltimes. The 8 μL [¹⁸F]fluoride mixture was gradually evaporated on thededicated EWOD heater 100 at 105° C. and then heated for an additional 3minutes. Additional 8 μL of the [¹⁸F]fluoride mixture was loaded andevaporated in the same manner as previously described. The [¹⁸F]fluoridemixture were dried via azeotropic distillation by transporting five MeCNdroplets (3 μL) through a first loading position 60 into the heater 100site and heated at 105° C. for 3 minutes. This drying step was repeated2 additional times. Two droplets of mannose triflate in DMSO (2 μL; 104mM) were pipetted onto the EWOD chip 98 through a second loadingposition 60 to avoid cross-contamination. Six MeCN droplets (3 μL) werethen loaded and transported to the heater 100 to initiate thefluorination reaction. The reaction droplet was gradually heated fromroom temperature to 120° C. over a period of 15 minutes. Four dropletsof HCl (2.5 μL; 1 N) were added to the crude [¹⁸F]FTAG droplet and thecombination was transported to the heater 100 region to perform thehydrolysis reaction. The reaction droplet was heated at 90° C. for 10mins. After the synthesis, the cover plate 58 was removed and the crude[¹⁸F]FDG product was extracted using 30 μL of H₂O for radio-TLC analysisand cartridge purification. The crude [¹⁸F]FDG was spotted onto a TLCsilica plate and was developed in 95:5 MeCN/H₂O solvent mixture. Thepercent composition of [¹⁸F]FDG and the unreacted [¹⁸F]fluoride wereanalyzed using a radio-TLC (MiniGITA star, Raytest). The conversionefficiency of the radiolabeling was analyzed to be 46% and thehydrolysis efficiency was 100%.

Cartridge Purification: The crude [¹⁸F]FDG microdroplet 20 synthesizedon the EWOD microfluidic chip 98 was purified using a purificationcartridge modified to avoid dilution of the final end product used inmicro-PET imaging. Commercially available purification cartridgesconsist of 280 mg to several grams of resin suited for macroscalesynthesis with several milliliters of sample volumes and therefore arenot designed for the microvolumes generated in the above procedure.Single dosage PET probes synthesized on the EWOD chip consisted ofseveral microliters of crude sample, which would be lost within thelarge amount of resin in a standard cartridge. Therefore, custom-madepurification cartridges were prepared by packing AG-50W-X4 (4 mg) andAG11 A8 (4 mg) resins with 50-100 mesh size (BioRad Laboratories) andneutral alumina (10 mg) with particle size 50-300 μm (Waters) within a3.175 mm ID polyurethane tubing. The resins were sandwiched between 2polyethylene frits (20 μm pore size) that were fitted with barb-to-lueradapters. The cartridge was first conditioned with water (1 mL; 18 MΩ)by gravity drip. The 30 μL crude product in water was pipetted andpassed through the cartridge by applying pressure using a 1 mL syringe.30 μL of water was then used to flush the cartridge. The sample eluentthat was collected after the first cartridge purification was spotted ona TLC plate and the radiochemical purity was found to be 85% byradio-TLC. The [¹⁸F]FDG mixture was therefore passed through a secondminiaturized cartridge packed with only neutral alumina (50 g) in thesimilar manner as previously described. The chemical purity after thesecond cartridge purification was analyzed using the radio-TLC and foundto be 99% (FIG. 29).

The purified product was then injected into mice for microPET imagingwith resulting images matching images taken from mice injected with[¹⁸F]FDG produced by conventional means.

EXAMPLE 4

DMSO is not commonly used in the macroscale radiosynthesis of [¹⁸F]FDGdue to the low vapor pressure of DMSO and the difficulty of removal.High-temperatures (which can damage the tracer) and long times (whichresults in substantial radioactive decay) are needed when usingevaporative removal. A cartridge can also be used, but results insubstantial increase in final volume which can be a problem especiallyfor preclinical imaging. On the other hand DMSO provides high solubilityof a wide range of organic compounds, high polarizability and theability to perform chemical reactions at higher temperature (due to itshigh boiling point). Using an EWOD chip 98 incorporating features of theinvention set forth herein, reliable radiofluorination of mannosetriflate with no-carrier-added [¹⁸F]fluoride in DMSO followed by acidhydrolysis, has been successfully demonstrated for the production of[¹⁸F]FDG. In micro-droplet radiosynthesis, in which 2-15 μL of DMSO isused during a typical radiofluorination reaction, the majority of theDMSO is evaporated at 120° C. after 10 minutes of the reaction.Subsequently, acid was added to the crude intermediate to perform thehydrolysis reaction to yield [¹⁸F]FDG with quantitative hydrolysisefficiencies. This result showed that the residual DMSO in the reactiondroplet did not affect the hydrolysis reaction nor the micro-PET imagingof a mouse.

EXAMPLE 5

To demonstrate the utility of the process and the EWOD chips 98 forchemical reactions other than radiochemistry, a simple chemicalsynthesis (not involving radioisotope) in a volatile organic solvent wasperformed using an EWOD chip. Due to the “open” structure of an EWODchip, microliter droplets of organic solvents evaporate very rapidly,especially at elevated temperatures, before chemical reactions canoccur. An approach utilized to allow the reactions to progress beforethe solvent is evaporated was to continually replenish the reaction sitewith new solvent droplets using the electrowetting mechanism totransport microliter volumes of solvent from a loading site 60 to thereaction site 100. A modified etherification reaction was performedbetween 1-naphtol and dibromobutane in acetonitrile (MeCN; bp. 82° C.)to yield 1-(4-bromobutoxy) naphthalene. The reaction is shownschematically in FIG. 30. In this chemical synthesis, 1-napthol (74 mM;2 μL) was first allowed to be reacted with K₂CO₃ (250 mM; 1 μL). in MeCNat 60° C. for 3 minutes. MeCN was continuously added to the reactiondroplet to maintain a liquid phase reaction. Subsequently, dibromobutanesolution in MeCN (3.7 M; 1 μL) was added to the reaction site and thesubstitution reaction was performed at 60° C. for 5 minutes.

After 5 minutes, the cover plate was removed, and the crude product wasextracted using MeCN. The crude product, labeled as P, was spotted ontoa TLC plate along with the starting material (labeled as S; 1-napthol)(FIG. 31). The TLC plate was developed in a solvent chamber containingdichloromethane/hexane (2:1 v/v) solvent mixture. The progress of thereaction was determined via tracking of the TLC stains using the UVlight. Based on the TLC analysis, the substitution reaction on the EWODchip yielded 1-(4-bromobutoxy)naphthalene, which appeared as a new spotwith R_(f) value of 0.9, while the unreacted 1-napthol had an R_(f)value of 0.2.

We claim:
 1. A method of performing radiosynthesis comprising: providingan electro-wetting-on-dielectric device (EWOD) comprising: a firstsubstrate having one or more electrically defined fluid paths along afirst surface thereof, each fluid path comprising discrete electrodepads, wherein adjacent electrode pads are separated from each other bynon-conductive spaces, each of the electrode pads having electricallyconductive lines attached thereto, each conductive line provided todeliver electrical signals to the conductive pad to which the line isattached to provide electrically directed movement of one or more fluiddroplets along the fluid paths; one or more fluid delivery sites locatedon or adjacent to the one or more electrically defined fluid paths; oneor more process operation sites located along the electrically definedfluid paths on the first substrate; delivering one or more fluiddroplets comprising a radioisotope in a liquid carrier to one of theprocess operation sites; delivering one or more fluid dropletscomprising at least one radiosynthesis reagent to one of the fluiddelivery sites; providing an electric field in a serial manner to theconductive pads adjacent to the fluid delivery sites to move the one ormore fluid droplets comprising the at least one radiosynthesis reagentto the process operation site with the radioisotope; reacting at leastone of the radiosynthesis reagents with the radioisotope at the processoperation site; and recovering the reaction product from the EWODdevice.
 2. The method of claim 1, wherein the reacting process comprisesmixing of the one or more fluid droplets comprising the radioisotopewith one or more fluid droplets comprising the at least oneradiosynthesis reagent.
 3. The method of claim 1, wherein the reactingprocess comprises heating of the one or more fluid droplets comprisingthe radioisotope with one or more fluid droplets comprising the at leastone radiosynthesis reagent.
 4. The method of claim 1, wherein the one ormore fluid droplets comprising the radioisotope are evaporated prior toreacting with the at least one radiosynthesis reagent.
 5. The method ofclaim 1, wherein the at least one radiosynthesis reagent comprises aprecursor.
 6. The method of claim 1, wherein the one or more fluiddroplets comprising the radioisotope and the one or more fluid dropletscomprising the at least one radiosynthesis reagent comprise volumeswithin of less than 1 mL.
 7. The method of claim 1, wherein the processoperation site comprises a heater.
 8. The method of claim 1, wherein thereaction product comprises a Fluorine-18 containing molecule.
 9. Amethod of performing Fluorine-18 radiosynthesis comprising: providing anelectro-wetting-on-dielectric device (EWOD) comprising: a firstsubstrate having one or more electrically defined fluid paths along afirst surface thereof, each fluid path comprising discrete electrodepads, wherein adjacent electrode pads are separated from each other bynon-conductive spaces, each of the electrode pads having electricallyconductive lines attached thereto, each conductive line provided todeliver electrical signals to the conductive pad to which the line isattached to provide electrically directed movement of one or more fluiddroplets along the fluid paths; one or more fluid delivery sites locatedon or adjacent to the one or more electrically defined fluid paths; oneor more process operation sites located along the electrically definedfluid paths on the first substrate; delivering one or more fluiddroplets comprising fluoride in a liquid carrier to one or more of theprocess operation sites; evaporating the one or more fluid dropletscomprising fluoride at the one or more process operation sites;delivering one or more fluid droplets comprising a precursor containingin an organic liquid carrier at one of the fluid delivery sites;providing an electric field in a serial manner to the conductive padsadjacent to the fluid delivery sites to move the one or more fluiddroplets comprising the precursor to the one or more process operationsites containing the evaporated fluoride; heating the one or moreprocess operation sites containing the evaporated fluoride and theprecursor to generate a reaction product; and recovering the reactionproduct from the EWOD device.
 10. The method of claim 1, whereinevaporating the one or more fluid droplets comprising fluoride at theone or more one or more process operation sites comprises adding aplurality of droplets of an anhydrous organic solvent while the one ormore heater sites are actively heating the one or more fluid dropletscomprising fluoride.
 11. The method of claim 10, wherein the anhydrousorganic solvent comprises anhydrous acetonitrile.
 12. The method ofclaim 9, further comprising: placing one or more fluid dropletscomprising a hydrolysis agent at one of the fluid delivery sites;providing an electric field in a serial manner to the conductive padsadjacent to the fluid delivery sites to move the one or more fluiddroplets comprising the hydrolysis agent to the heater sites containingthe reaction product.
 13. The method of claim 9, wherein the precursorcomprises mannose triflate.
 14. A method of performing radiosynthesiscomprising: providing an electro-wetting-on-dielectric device (EWOD)comprising: a first substrate having one or more electrically definedfluid paths along a first surface thereof, each fluid path comprisingdiscrete electrode pads, wherein adjacent electrode pads are separatedfrom each other by non-conductive spaces, each of the electrode padshaving electrically conductive lines attached thereto, each conductiveline provided to deliver electrical signals to the conductive pad towhich the line is attached to provide electrically directed movement ofone or more fluid droplets along the fluid paths; one or more fluiddelivery sites located on or adjacent to the one or more electricallydefined fluid paths; one or more process operation sites located alongthe electrically defined fluid paths on the first substrate; deliveringone or more fluid droplets comprising at least one radiosynthesisreagent to one of the process operation sites; delivering one or morefluid droplets comprising a radioisotope in a liquid carrier to one ofthe fluid delivery sites, the delivery site being spaced from the one ormore operation sites; providing an electric field in a serial manner tothe conductive pads adjacent to the fluid delivery sites to move the oneor more fluid droplets comprising the radioisotope to the processoperation site containing the at least one radiosynthesis reagent;reacting at least one of the radiosynthesis reagents with theradioisotope at the process operation site; and recovering the reactionproduct from the EWOD device.
 15. The method of claim 14, wherein thereacting process comprises mixing of the one or more fluid dropletscomprising the radioisotope with one or more fluid droplets comprisingthe at least one radiosynthesis reagent.
 16. The method of claim 14,wherein the reacting process comprises heating of the one or more fluiddroplets comprising the radioisotope with one or more fluid dropletscomprising the at least one radiosynthesis reagent.